There are a number of different circumstances in which it is desirable to perform an analysis to identify and/or measure compounds in a sample. Such samples may be taken directly from the environment or they may be provided by front end specialized devices to separate or prepare compounds before analysis.
Differential Mobility Spectrometry (DMS), also referred to as High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) and Field Ion Spectrometry (FIS), are technologies for gas phase ion sample separation and analysis. Researchers have interfaced DMS with mass spectrometry to take advantage of the atmospheric pressure, gas phase, and continuous ion separation capabilities of DMS and the detection specificity offered by mass spectrometry.
By interfacing DMS with mass spectrometry, researchers have demonstrated benefits in numerous areas of sample analysis, including proteomics, peptide/protein conformation, pharmacokinetic, and metabolism analysis. In addition to pharmaceutical/biotech applications, DMS has been incorporated into products designed for trace level explosives detection as well as petroleum monitoring. Despite the demonstrated success of the technology, ion behavior during the differential mobility separation is not well understood for the wide range of analytes that are being analyzed by this technology. Accordingly, there is a need to enhance separation efficiency and enable predictable separation controls for a differential mobility spectrometer (DMS) interfacing with a mass spectrometer (MS).
DMS can be viewed as a spin off from conventional ion mobility spectrometry (IMS). In conventional IMS, ions are pulsed into and then pulled through a flight tube by a constant electric field. The ions interact with a bath gas in the flight tube and the interactions affect the time it takes an ion to pass through the flight tube. Unlike time of flight (TOF) MS where an ion's time through a flight tube is based solely on its mass to charge ratio (due to collision free travel in a vacuum), ions in conventional IMS are not separated in a vacuum, enabling interactions with the bath gas molecules. These interactions are specific for each analyte ion of a sample, leading to an ion separation based on more than just mass/charge ratio.
DMS is similar to conventional IMS in that the ions are separated in a bath or drift gas. However, typically, an asymmetric electric field waveform is applied to two parallel electrode plates through which the ions pass in a continuous, non-pulsed, manner. The electrical waveform consists of a high field duration of one polarity and then a low field duration of opposite polarity. The duration of the high field and weak field portions are applied such that the net voltage being applied to the electrode is zero. FIG. 1 includes an illustration 100 of the high and low voltages of opposite polarity applied to generate the asymmetric electrical waveform (identified as an Rf (field) voltage, correlating to the high voltage value) and a conceptual diagram of a DMS filter 102 where the path of an ion M+ is subject to an asymmetric field resulting from the asymmetric waveform. As can be seen in illustration 100, after one cycle of the waveform, the net voltage applied to the DMS filter electrode is zero.
However, the ion's mobility in this asymmetric electric field demonstrates a net movement towards the bottom electrode plate of the DMS filter 102. This example shows that, in DMS, an ion's mobility is not proportional under the influence of a low electric field compared to a high electric field. Since an ion may experience a net mobility towards one of the electrode plates during its travel between the plates, a compensation voltage (Vc) is applied to maintain a safe trajectory for the ion through the DMS filter 102 plates without striking them. The ions are passed between the two electrodes by either being pushed through with a pressurized gas flow upstream of the electrode plates or pulled through by a pump downstream from the electrodes.
In conventional IMS, as well as DMS, ions are separated in a gas at pressures sufficient for the occurrence of collisions between ions and the neutral gas molecules. The smaller the ion, the fewer collisions it will experience as it is pulled through the drift gas. Because of this, an ion's cross sectional area may play a significant role in it's mobility through the drift gas. As shown in FIG. 1, an ion's mobility is not proportional under the influence of a low electric field compared to a high electric field. This difference in mobility may be related to clustering/de-clustering reactions taking place as an ion experiences the weak and strong electric fields. An ion experiences clustering with neutral molecules in the drift gas during the weak field portion of the waveform, resulting in an increased cross sectional area. During the strong field portion of the waveform, the cluster may be dissociated, reducing the ion's cross sectional area.
Recently, DMS research has focused on understanding the gas phase molecular interactions taking place and how they influence an ions' mobility in the DMS sensor. Existing FAIMS-MS systems have demonstrated DMS separation between certain monomer ions and non-covalently bound cluster/dimer ions. These systems have provided evidence that non-covalently bound dimer/cluster ions can have different differential mobility behavior from their monomer counterparts. This indicates that the cluster/dimer ions were created prior to entering the DMS sensor, and were not dissociated back to their monomer counterparts upon entering the asymmetric electric field of the sensor and/or DMS filter. The formation of these cluster/dimer ions may effect the detection of ions of interest within a sample analysis system such as a DMS-MS. Accordingly, there is a need for compensating for and/or accounting for the presence of cluster/dimer ions and other compounds that result from gas phase molecular interactions in a sample analysis system to enhance the accuracy and resolution of these systems.
Existing DMS-based systems have analyzed sample ions through the use of various vapor modified drift gases for which the proposed process is via clustering/de-clustering interactions between a monomer analyte ion and neutral drift gas modifier/dopant molecule in which the analyte ion's effective cross sectional area is changed. While existing DMS-based systems have shown a change in an analyte ion's differential mobility behavior through the use of drift gas modifiers or dopants, there remains a need for a clear model with regards to the underlying interactions between the modifier and analyte, and the mechanism(s) by which those interactions change an analyte ion's differential mobility behavior.
By employing a DMS as a pre-filter to a MS, existing FAIMS-MS and/or DMS-MS systems have increased the detection sensitivity and resolution of sample analysis by reducing the amount of contaminants or unwanted particles that interact with the ions of interest in a MS. Electrospray ionization (ESI) has been employed with FAIMS-MS to facilitate the analysis of certain liquid samples. However, direct infusion of samples using ESI has typically been avoided, particularly with complex samples, because of problems with competitive ion suppression. Competitive ion suppression has limited the accuracy of existing ESI-FAIMS-MS systems by reducing the quantity of ions of interest that are eventually detected in the MS. Because of ion suppression, analyte separation techniques prior to ESI, such as Liquid Chromatography (LC), Gas Chromatography (GC), and Capillary Electrophoresis (CE), have been utilized to minimize ion suppression effects. Accordingly, there is a need for providing an ESI-DMS-MS system having enhanced capabilities that reduce competitive ion suppression and/or compensate for the effects of such suppression when quantizing certain ion species.
It has long been known that the conformational structure of a protein has an effect on a wide spectrum of its biological activities including the control of signaling pathways, biosynthesis, its interaction with binding ligands, the transport of drugs and other chemical agents. In addition to equilibrium related intermediates, there has also been interest in the so-called transient folding states which represent a highly dynamic system that may play an important role in biological functions. Traditionally, nuclear magnetic resonance (NMR) has been the common instrumental technique for monitoring these physical transformations in solution. However, the development of electrospray ionization (ESI) techniques has introduced new opportunities for studying protein conformational features by mass spectrometry. Since conformational change effectively reflects a change in the volume of space occupied by the protein, and thus its cross sectional area, ion mobility mass spectrometry provides an attractive complementary approach for the study of such processes. Two basic mass spectrometric approaches are typically used to examine variations in the conformational structure of a protein:                (i) shift of the charge-state distribution pattern during electrospray ionization and,        (ii) hydrogen/deuterium (H/D) exchange.        
For example, it has long been known that, upon electrospray ionization, a folded (spherically shaped) protein is able to accommodate fewer positive charges on its surface due to increased coulombic repulsion and/or due to reduced exposure of possible protonation sites of basic amino acids. Significantly, if two conformations co-exist in the same solution, the ESI spectrum may exhibit a bimodal pattern representative of the isomeric mixture. In turn, the folding status is also known to influence the exchange of highly labile (amino or carboxylic) hydrogens with deuterium made available through the solvent medium, and the resulting mass increments are used to, at least qualitatively, establish the occurrence of conformational changes.
In general, simple H/D exchange is not sufficient to identify specific amino acids in the sequence of the protein that may have undergone exchange without enzymatic digestion of the protein and amino acid sequencing of the resulting peptides. For highly labile hydrogens, back exchange is also a major concern. However, H/D may provide some guide toward the identification of protected regions in conformers. It has been demonstrated that the two conformers of DvHY64A, a mutated form of Cytochrome C553, exhibited different levels of H/D exchange indicating that there is a sub-population of amide hydrogens which remain shielded because the folded forms of the two conformers are perfectly stable.
There are several examples of the use of the two aforementioned approaches in conjunction with combined IMS-MS to study protein conformations (See Valentine, et al., H/D Exchange Levels of Shape-Resolved Cytochrome c Conformers in the Gas Phase, J. Am. Chem. Soc., 1997, 119, 3558-3566). Valentine et al. showed that variation of the voltage at which protein ions are injected into the drift tube of an IMS resulted in the formation of different conformers. Lower deuterium incorporation was observed for the charge states of compact conformers as opposed to the unfolded forms and, consistent with the discussion presented above, an analogous trend was observed for the respective charge state distributions. More recently, a tandem IMS-IMS-MS combination has been proposed in which the first IMS selects specific ions which are subjected to collision activated dissociation (CAD) and the fragments transferred to the second drift tube for further separation and CAD followed by MS analysis. (See Koeniger et al., An IMS-IMS Analogue of MS-MS, 2006, 78, 4161-4174).
In addition to conventional drift tube IMS, the capability of FAIMS or DMS for the study of protein conformations has been explored. Conformers of ubiquitin were generated in solution at set pH values and introduced into the FAIMS-MS system by electrospray ionization. Separation in the FAIMS analyzer was found to be dependent on the structure (conformation) of a given protein ion and several conformers of the same charge state were often resolved in the DMS. Moreover, samples sprayed from solutions of different pH yielded different charge distribution patterns as well as different conformers as indicated from the shifts in the Vc spectra. Of particular significance was the conformer-specific distribution of Na+ and other adduct ions, leading to the conclusion that the addition of “spectator” ions to form protein-spectator adducts may lead to information about the conformational structure of the protein.
Related research has investigated the electrospray mass spectra of the amyloidogenic protein β2-microglobulin (β2m) using a FAIMS-MS combination over a range of several pH units. As expected, at each pH, a different charge distribution envelope was observed. However, when the different charge states were further screened by combining the Vc and MS spectra, it was determined that each charge state reflected the presence of multiple conformers in the sprayed solution which could then be resolved in the FAIMS filter. (See Borysik et al., Separation of β2-microglobulin conformers by high-field asymmetric waveform ion mobility mass spectrometry (FAIMS) coupled to electrospray ionization mass spectrometry, RCMS, 2004, 18, 2229-2234).